Extract from indigo naturalis and a process for preparing the same

ABSTRACT

The present invention relates to a process for preparing an extract from one or more botanical raw materials, such as Indigo Naturalis and the extract itself. The present invention also relates to a composition comprising the extract, as well as the use of composition in medical or cosmetic applications.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application claiming priority under 35U.S.C. § 120 to U.S. patent application Ser. No. 15/592,893 filed May11, 2017, which claims the benefit, under 35 U.S.C. § 365 ofInternational Application PCT/EP2016/057761 filed Apr. 8, 2016, whichwas published in the English language on Oct. 13, 2016, underInternational Publication No. WO/2016/162484 A1, and which claims thebenefit of European patent application No. 15163060.5, filed Apr. 9,2015, the disclosure of each of these prior applications is herebyincorporated by reference herein in its entirety.

TECHNICAL FIELD

The present invention relates to a process for preparing an extract fromone or more botanical raw materials, such as Indigo Naturalis and theextract itself. The present invention also relates to a compositioncomprising the extract, as well as the use of composition in medical orcosmetic applications.

BACKGROUND

The conventional treatments for psoriasis are generally designedaccording to the age, gender, occupation and cognitive ability of apatient, the types and distribution of lesions, patient's response(s) toprevious therapeutic method(s), and other medical histories of thepatient. The primary therapeutic methods for psoriasis include topicaltherapy, systemic therapy, injection of biologics and phototherapy.Compositions for topical therapy include, e.g., corticosteroids,anthralin (available as Margiton®), coal tar (available as Polytar®),calcitriol (available as Silkis®), tazarotene (available as Tazorac®),salicylic acid, and these compositions are suitable for treatingpsoriasis patients with mild symptoms. Oral preparations of e.g.,methotrexate (MTX), cyclosporine, and retinoids are commonly used forsystemic therapy and are suitable for treating psoriasis patients withmedium to severe symptoms. Biologics include alefacept (available asAmevive®), efalizumab (available as Raptiva®), etanercept (available asEnbrel®) and adalimumab (available as Humira®), and they are suited forinjecting into psoriasis patients with medium to severe symptoms.Phototherapy, e.g., ultraviolet B (UVB) phototherapy, photochemotherapysuch as psoralen plus ultraviolet A (PUVA), is suitable for treatingpsoriasis patients with severe symptoms.

However, additional therapeutic methods are desirable.

SUMMARY

Indigo Naturalis, for example Qingdai, is a dark-blue powder preparedfrom leaves of Indigo-bearing plants or Indigo-producing plants. Saidplants are preferably selected from the group consisting of Indigoferatinctoria L., Baphicacanthus cusia (Nees) Bremek (syn. Strobilanthescusia (Nees), Persicaria tinctoria (Aiton) Spach. (syn. Polygonumtinctorium Aiton, P. tinctorium Lour.) and Isatis tinctoria L. (syn.Isatis indigotica Fort.).

One of the drawbacks encountered with the use of Indigo Naturalis orQingdai is the dark color it produces on the skin and it leaves stainson clothes. Qingdai is the current name for Indigo Naturalis. It isextracted from Indigo-bearing or Indigo-producing plants with a NaOH orKOH aqueous solution and corresponds to a mixture of around 5-15%organic compounds including alkaloids among which indigo and indirubinare present, and 85-95% inorganic compounds such as calcium carbonateand calcium hydroxide.

Another drawback may be that traditionally, only indigo and indirubinare controlled in Indigo Naturalis product.

Thus, there remains an unmet need to develop an extract from IndigoNaturalis or from Indigo-bearing or Indigo-producing plants into amarketable drug, to obtain an extract less colored than Indigo Naturalisand to characterize and control the majority of the components of suchextract. More specifically, it would be advantageous to provide anextract less colored while maintaining or increasing its efficacy fortreatment or alleviation of skin diseases or disorders such aspsoriasis.

The present invention solves this problem by providing with aneasy-to-handle refined extract retaining active components from IndigoNaturalis or Qingdai to ensure clinical efficacy and safety.

The present invention, in part, deals with a process for obtaining anextract from Indigo Naturalis, for example from Qingdai, that in turncontains e.g., indirubin and indigo for treatment or alleviation of skindiseases or disorders such as psoriasis.

In one aspect, the present invention provides a process for preparing anextract from one or more botanical raw materials, such as IndigoNaturalis or the leaves and/or stems of one or more Indigo-bearingplants or indigo-producing plants, preferably selected from the groupconsisting of Indigofera tinctoria L., Baphicacanthus cusia (Nees)Bremek (syn. Strobilanthes cusia (Nees), Persicaria tinctoria (Aiton)Spach. (syn. Polygonum tinctorium Aiton, P. tinctorium Lour.) and Isatistinctoria L. (syn. Isatis indigotica Fort.). The process comprises thefollowing steps:

-   -   a) an extraction step: extracting Indigo Naturalis or the leaves        and/or stems of one or more plants as selected above with a        first polar solvent or moderately polar solvent to obtain a        mixture of extraction;    -   b) a filtration step: filtering the mixture of extraction to        obtain a filtrate;    -   c) a concentration step: concentrating the filtrate to obtain a        crude extract;    -   d) a washing step: washing the crude extract with a non-polar        solvent, and optionally a second polar solvent, to obtain a        washing mixture; and    -   e) a filtration step: filtering the washing mixture to obtain a        refined extract optionally after a drying step, for example,        according to a conventional method for drying.

The process according to the invention allows obtaining a refinedextract; such a refined extract is as defined below. In the followingdescription, the term “botanical raw material” refers to IndigoNaturalis (for example, commercially available Qingdai), or leavesand/or stems of an Indigo-bearing plant or Indigo-producing plant,preferably selected from the group consisting of Indigofera tinctoriaL., Baphicacanthus cusia (Nees) Bremek (syn. Strobilanthes cusia (Nees),Persicaria tinctoria (Aiton) Spach. (syn. Polygonum tinctorium Aiton, P.tinctorium Lour.) and Isatis tinctoria L. (syn. Isatis indigotica Fort),after harvest and collection, may be processed by, for example,fermentation.

In some embodiments, the solvent used in the extraction step a) mayinclude, but is not limited to, dimethyl formamide (DMF), ethyl acetate,ethanol (including aqueous ethanol such as, for example, 85% ethanol),dimethylsulfoxide (DMSO), dichloromethane (DCM), tetrahydrofuran (THF),dimethylacetamide (DMA), acetone, 2-butanone, acetonitrile, isopropylacetate, 2-methyl tetrahydrofurane (MeTHF), methyl tert-butyl ether,water, methanol, chloroform, terpene (limonene, p-cimene, etc.), or acombination thereof. Preferably, the solvent can be ethyl acetate,MeTHF, ethanol or aqueous ethanol.

Further, the extraction step a) can be conducted at a ratio of botanicalraw material to solvent (i.e., the gram of the material to be extractedto the mL of the solvent) in a range from 1:5 to 1:150 (g/mL), forexample, in a range from 1:5 to 1:100, for example, 1:6, 1:15, 1:20,1:30, 1:50, 1:100.

In a particular embodiment, the solvent used in the extraction step a)is an organic solvent. More preferably, the solvent is selected from thegroup consisting of dimethylformamide, ethyl acetate, ethanol,dimethylsulfoxide, dichloromethane, tetrahydrofuran, dimethylacetamide,acetone, 2-butanone, acetonitrile, isopropyl acetate, 2-methyltetrahydrofuran, methyl tert-butyl ether, methanol, chloroform, terpeneand a combination thereof.

In some embodiments, the extraction step a) can be repeated until thereis less than 0.10% of indirubin content (% w/w) in the remainingbotanical raw material filtered at the filtration step b) as measured bythe HPLC method disclosed in Example 8A.

In some other embodiments, the extraction step a) is performed byheating, for example till reflux.

The extraction step a) is followed by a filtration step b), and then aconcentration step c) for concentrating the filtrate to provide a crudeextract. The filtration step b) can be performed while hot.

In some embodiments, the washing step d) can be repeated until theamount of indirubin (% w/w) in the refined extract is at least 55% (w/w)as measured by the HPLC method disclosed in Example 8A.

In some embodiments, the solvent used in washing step d) is selectedfrom the group comprising water, an alkane with 5 to 8 carbon atoms, analcohol with 2 to 6 carbon atoms and a combination thereof. For example,the solvent includes, but is not limited to, hexane, heptane, ethanol,water or a combination thereof. For example, in the washing step d),hexane or heptane, and aqueous ethanol are used in sequence orsimultaneously in the case that two or more of the solvents are used.

According to an embodiment, the non-polar solvent used in step d) ishexane.

According to another specific embodiment, step d) is carried out with anon-polar solvent and a second polar solvent. Preferably, the secondpolar solvent is ethanol. More preferably, the non-polar solvent used instep d) is hexane and the second polar solvent is ethanol.

In some other embodiments, a crude extract obtained from theconcentration step c) is subjected to the following procedure for atleast one cycle till obtaining a refined extract: the crude extract iswashed by a solvent (step d)), and filtered (step (e)) to yield arefined extract, optionally followed by a drying step. According to aspecific embodiment, the washing step d) and filtration step e) areperformed by only one cycle to obtain the refined extract. When morethan one cycle is applied, the same or different solvents for washingcan be used. Further, the crude extract can be washed with a solventunder reflux, the mixture can be cooled to room temperature and thenfiltered to yield a refined extract, optionally followed by a dryingstep.

In a preferred embodiment, two cycles are performed. Particularly, thecrude extract obtained by the concentration step c) is washed in anon-polar solvent, preferably hexane (step d) and filtered (step e),optionally followed by a drying step. The hexane extract is then washedby an organic polar solvent, preferably ethanol (step d) and thenfiltered (step e) to obtain a refined extract, optionally followed by adrying step.

Optionally, a micronization step is performed after step e), providingthereby a refined extract having a particle size between 25 and 35 μm,preferably of about 30 μm.

In another preferred embodiment, when the refined extract is micronizedin the last step, 99% of the obtained particles are less or equal to 30μm.

In another aspect, the present invention provides a refined extract fromIndigo Naturalis.

The present invention also provides a refined extract containingindirubin as a component in an amount of at least 55% (w/w) of therefined extract. In some embodiments, indirubin is present in an amountof at least 60%, 65%, 70%, 75%, 80% or 85% (w/w) of the refined extract.For example, the indirubin can be in an amount of 55-90% (w/w) of therefined extract, e.g., 55-60%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%,55-90%, 60-65%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 65-70%, 65-75%,65-80%, 65-85%, 65-90%, 70-75%, 70-80%, 70-85%, 70-90%, 75-80%, 75-85%,75-90%, 80-85%, 80-90%, or 85-90% (w/w) of the refined extract.

Preferably, the present invention provides a refined extract containingindirubin as a component in an amount of at least 65% (w/w) of therefined extract.

Preferably, the indirubin is in an amount of 65-90% (w/w) of the refinedextract.

In some embodiments, the refined extract contains indigo as a component.Indigo can be in an amount of 0-15% (w/w) of the refined extract, forexample, 0.1-15%, 0.5-15%, 1-15%, 2-15%, or 3-15% (w/w) of the refinedextract.

In some other embodiments, the refined extract further containstryptanthrin as a component. Tryptanthrin can be in an amount of 0.01-5%or preferably 0.1-5% (w/w) of the refined extract, for example, 0.01-1%,0.05-1%, 0.1-1%, 0.1-5%, 0.5-1%, or 0.5-5% (w/w) of the refined extract.

In further some embodiments, the refined extract further containsqingdainone as a component. Qingdainone can be in an amount of 0.1-5%(w/w) of the refined extract, for example, 0.1-1%, 0.1-5%, 0.5-1%, or0.5-5% (w/w) of the refined extract.

In further some other embodiments, in the refined extract, comprisingtwo or more of those described above, represents at least 60% (w/w) ofthe refined extract, for example 65%, 70%, 75%, 80%, 85%, 90%, or 95%(w/w), and preferably at least 90% (w/w). The characterized components,comprising two or more of those described above, are in an amount of60-99% (w/w) of the refined extract, for example 60-95%, 65-95%, 70-95%,80-95%, 85-95%, 90-95%, 60-99%, 65-99%, 70-99%, 80-99%, 85-99% or 90-99%(w/w) and preferably in an amount of 90-99% (w/w).

In further another aspect, the present invention provides apharmaceutical composition comprising a refined extract as describedabove, and a pharmaceutically acceptable carrier. The composition is inthe form for topical or oral administration.

In further another aspect, the present invention provides a cosmeticcomposition comprising a refined extract as described above, and acosmetically acceptable carrier. The composition is in the form fortopical administration.

In still another aspect, the present invention provides a use of therefined extract in the preparation of a pharmaceutical or cosmeticcomposition for inhibiting proliferation or differentiation ofkeratinocytes, inhibiting infiltration of mononuclear cells into thedermis and epidermis, inhibiting vascular alteration resulting inhyperplastic blood vessels, or inhibiting up-regulation of adhesionmolecules on endothelia cells. The use comprises administering thejust-described composition to a subject (e.g. human) in need thereof.

In further another aspect, the present invention provides anafore-described composition for the treatment or alleviation of a skindisease or condition selected from the group consisting of psoriasis,inflammatory skin conditions, onychomycosis, skin cancer, abnormalkeratinization induced diseases, skin aging, pustulardermatosis andCutaneous T Cell Lymphoma (CTCL).

In another aspect, the present invention provides a method forinhibiting proliferation or differentiation of keratinocytes, inhibitinginfiltration of mononuclear cells into the dermis and epidermis,inhibiting vascular alteration resulting in hyperplastic blood vessels,or inhibiting up-regulation of adhesion molecules on endothelia cells,which comprises a step of contacting the refined extract of the presentinvention to a cell in need thereof. This method may be applied in vitroor in vivo.

In still further another aspect, the present invention provides a methodfor the treatment of skin disease or condition comprising administeringan effective amount of the refined extract according to the presentinvention to subjects in need thereof. The disease or condition isselected from the group consisting of psoriasis, inflammatory skinconditions, onychomycosis, skin cancer, abnormal keratinization induceddiseases, skin aging, pustulardermatosis and CTCL.

In some embodiments, the inflammatory skin conditions can be atopicdermatitis, eczema or superinfected skin. The skin aging can be skinrejuvenation, tissue regeneration for scars or skin senescence. Theabnormal keratinization can be acne, ichtyosis or palmoplantarkeratoderma. The skin cancer can be precancerous skin cancer, forexample, Actinic Keratosis (AK), Bowen's disease; skin cancer, forexample, SCC, BCC, NMSC; melanoma and HPV induced skin cancer.

DESCRIPTION OF DRAWINGS

The above and other features and advantages of the present inventionwill become apparent with reference to the following detaileddescription and the accompanying drawings.

FIGS. 1A and 1B: Illustrative HPLC chromatograms of indigo (FIG. 1A) andindirubin (FIG. 1B).

FIGS. 2A to 2C: Illustrative HPLC chromatograms of thryptanthrin (FIG.2A), Qingdai (FIG. 2B) and a refined extract of Example 2 (FIG. 2C).

FIG. 3: Group and dosing for in vivo evaluation of extracts from Qingdaiin IL-22 induced psoriasis model. Five studies were designed andoutlined in study 1-5, respectively. In Study 1, 100 ng and 500 ng ofIL-22 in 20 μL saline, i.d., were used for induction. In study 2-5, 500ng of IL-22 in 204 saline, i.d., were used for induction.

FIG. 4: Figures illustrate ear inflammation after intradermal injectionof IL-22. In the studies, ears of mice were injected intradermally andear thickness was measured on days between injections.

FIG. 5: Figures illustrate the effects of extracts from Qingdai on earinflammation after intradermal injection of IL-22.

DETAILED DESCRIPTION

As used herein, indirubin, indigo, tryptanthrin, and qingdainone havethe formulas as mentioned in the literature, and more particularly thefollowing structures.

A “polar solvent” refers to a solvent having a high dielectric constantand a high dipole moment; and examples include water, alcohols, forexample, alcohols from 2 to 6 carbon atoms (such as ethanol),acetonitrile, dimethylfomamide (DMF), and dimethylsulfoxide (DMSO).

A “moderately polar solvent” refers to a solvent having moderately highdielectric constant; and examples include ethyl acetate, dichloromethane(DCM), tetrahydrofuran (THF), dimethylacetamide (DMA), acetone,2-butanone, isopropyl acetate, 1,4-dioxane, 2-methyltetrahydrofuran(MeTHF), methyl tert-butyl ether (MTBE).

A “non-polar solvent” refers to a solvent having low dielectricconstant; and examples include diethyl ether, petroleum ether (PE),toluene and alkanes from 5 to 8 carbon atoms, for example, heptane,hexane, pentane or cyclohexane.

A “room temperature” refers to 18° C.-35° C.

The term “psoriasis” or “psoriasis disease” refers to all types ofpsoriasis well known to those skilled in the art. It includes, but isnot limited to, chronic plaque psoriasis, guttate psoriasis,erythrodermic psoriasis, pustular psoriasis, inverse psoriasis (alsoknown as flexural psoriasis), nail psoriasis, psoriatic arthritis.

The term “refined extract” refers to a solid, semi-solid or oilyextract, preferably solid extract, which contains less than 10% (w/w) ofwater and/or solvents used in the process for preparing the said refinedextract. A refined extract is more preferably characterized by anincrease amount of active ingredients, including alkaloids among whichindigo, indirubin, tryptanthrin, and/or qingdainone are present,preferably enriched in indirubin, compared to Qingdai or IndigoNaturalis. More specifically, the refined extract according to theinvention comprises at least 60%, or more preferably more than 70%,(w/w) of active ingredients, including indigo, indirubin, tryptanthrin,and/or qingdainone.

The term “crude extract”, as used herein, refers to a solid, semi-solidor oily extract, preferably solid or semi-solid extract, which containsless than 15% (w/w) (e.g., 5-15%, 5-10%) of water and/or solvents usedin the process for preparing the refined extract. The crude extract isless enriched in indirubin, than the refined extract as compared toQingdai or Indigo Naturalis. The crude extract is obtained by theconcentration step c) according to the invention. The concentration stepis more particularly carried out by sending the filtrate to aconcentrator (for instance at reduced pressure), as to remove waterand/or solvents used in the process and concentrating thereby the activeingredients present in the extract, including indigo, indirubin,tryptanthrin, and/or qingdainone.

“one cycle”, as used herein, refers to the two steps of the washing stepd) and filtration step e) which are performed sequentially once. “twocycles”, as used herein, refers to the two steps of the washing step d)and filtration step e) which are performed sequentially twice.

The term “an effective amount” refers to a dose level of the refinedextract which yields a therapeutic benefit (for example, amelioration,alleviation or cure of the diseases, disorders or symptoms) to a patienton average.

The present invention provides a process for preparing a refined extractfrom Indigo Naturalis or from the leaves and/or stems of one or moreIndigo-bearing plants or Indigo-producing plants, preferably selectedfrom the group consisting of Indigofera tinctoria L., Baphicacanthuscusia (Nees) Bremek (syn. Strobilanthes cusia (Nees), Persicariatinctoria (Aiton) Spach. (syn. Polygonum tinctorium Aiton, P. tinctoriumLour.) and Isatis tinctoria L. (syn. Isatis indigotica Fort.)

Indigo Naturalis is obtained from leaves and stems, preferably fromleaves, of Indigofera tinctoria L., Baphicacanthus cusia (Nees) Bremek(syn. Strobilanthes cusia (Nees), Persicaria tinctoria (Aiton) Spach.(syn. Polygonum tinctorium Aiton, P. tinctorium Lour.) and Isatistinctoria L. (syn. Isatis indigotica Fort.). Preferably, IndigoNaturalis is obtained from leaves and stems of Persicaria Tinctoriaand/or Baphicacanthus cusia.

While Indigo Naturalis is commercially available (examples of IndigoNaturalis commercially available (plant/supplier): Baphicacanthuscusia/Delong; Indigofera tinctoria/KMA exports or Sam Vegetable ColoursPVT Ltd; Isatis tinctoria/Andrea Primavera or Bleu de Lectoure;Polygonum tinctorium/CouleurGarance or EARL 4 saisons), it can beproduced from the leaves and stems of one or more of above plants bymethods commonly known in the art. These methods can be summarized asfollows: freshly harvested stems and leaves of Persicaria Tinctoriaand/or Baphicacanthus cusia are added to a pool in the open air, thewater is added to the pool to cover the stems/leaves. After soaking forfew days (26-30° C.), the stems/leaves will become rotten. Then limesoda is added while stirring. When the color of the soaking mixturechanged from green to deep violet, the precipitate is collected, washed(usually with water for 2-3 times), and then dried to yield IndigoNaturalis powder.

A refined extract may be prepared by a process according to theinvention comprising the following steps consisting of: a) (i) adding anextracting solvent, a polar or moderately polar solvent (such as analcohol or ethyl acetate), to Indigo Naturalis powder to yield amixture; (ii) heating and stirring the mixture for a period of time(e.g., 30 min, 1 hour, 2 hours); b) (iii) filtering the heated mixturewhile hot to remove insoluble by-products to yield a filtrate; c) (iv)concentrating the filtrate to yield a crude extract; d) (v) adding awashing solvent (for example, water, or a non-polar, or a polar solventor a mixture thereof) to the crude extract to yield a washing mixture;(vi) heating and stirring the washing mixture for a period of time(e.g., 30 min, 1 hour, 2 hours); e) (vii) filtering the washing mixture,for example at room temperature (e.g. 18° C.-35° C.) to collect arefined extract; optionally (viii) repeating steps (v) to (vii) untilthe amount of indirubin (% w/w) in the residue (i.e., the extract) ismore than 55% (w/w), preferably more than 65% (w/w), as measured by HPLCmethod disclosed in Example 8A, optionally (ix) drying the refinedextract according to a conventional method (e.g., air-drying,lyophilization). The washing solvent in steps (v) and (viii) can be thesame or different.

In a preferred embodiment, a refined extract is prepared by a processcomprising the steps of:

-   -   a) extracting Indigo Naturalis with ethanol at reflux between 2        and 8 hours,    -   b) filtering the mixture at a temperature not less than 65° C.        to obtain a filtrate,    -   c) concentrating the filtrate, to obtain a crude extract, said        crude extract is optionally filtered (with addition of water) in        order to remove completely the solvent and the last components        still present in the solvent and dried,    -   d) (i) washing the crude extract with hexane at a temperature        not less than 50° C. between 15 and 60 min,        -   (ii) filtering at room temperature the mixture obtained at            step d) (i) to obtain a product, optionally rinsing it with            ethanol and water        -   (iii) washing the product obtained at step d) (ii) with            ethanol at reflux, and        -   e) filtering at room temperature the washing mixture            obtained at step d) and drying the resulting product at a            temperature less than 80° C. to obtain an extract which is            optionally micronized.

In another preferred embodiment, when the refined extract is micronizedin the last step, the particle size is around 99% in the range 25 to 35μm, preferably of about 30 μm.

As used herein, “about” or “around” will be understood by a person ofordinary skill in the art and will vary to some extent on the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art given the context in which it isused, “about” or “around” will mean up to plus or minus 20%, preferably10% of the particular term.

The contents of ingredients such as indigo, indirubin, tryptanthrin andqingdainone can be quantitated by HPLC methods as disclosed in Example8.

The just described process makes it possible to obtain a refined extractthat is easy to handle and can be further formulated in pharmaceuticalor cosmetic compositions. Moreover, the process provides an extract withan enriched indirubin.

At least 60% (w/w) of the component of the refined extract thus obtainedare characterized; preferably at least 90% (w/w), among them one or moreare selected from the list consisting of indigo, indirubin, tryptanthrinand qingdainone. While the refined extract obtained from the process isin a solid form, a preparation of a liquid form for the refined extractmay be made based on handling or formulating needs by a simpledissolution or solubilization of the solid refined extract.

In this case, a liquid form for the refined extract may be prepared by aprocess according to the invention comprising the following stepsconsisting of: a) (i) adding an extracting solvent, a polar ormoderately polar solvent (such as an alcohol or ethyl acetate), toIndigo Naturalis powder to yield a mixture; (ii) heating and stirringthe mixture for a period of time (e.g., 30 min, 1 hour, 2 hours); b)(iii) filtering the heated mixture while hot to remove insolubleby-products to yield a filtrate; c) (iv) concentrating the filtrate toyield a crude extract; d) (v) adding a washing solvent (for example,water, or a non-polar, or a polar solvent, or a mixture thereof) to thecrude extract to yield a washing mixture; (vi) heating and stirring thewashing mixture for a period of time (e.g., 30 min, 1 hour, 2 hours); e)(vii) filtering the washing mixture, for example at room temperature(e.g. 18° C.-35° C.), to collect a refined extract; optionally (viii)repeating steps (v) to (vii) until the amount of indirubin (% w/w) inthe residue (i.e., the extract) is more than 55% (w/w), preferably morethan 65% (w/w), as measured by HPLC method disclosed in Example 8A, (ix)solubilizing the refined extract thus obtained in a pharmaceuticallyacceptable solvent or carrier. The washing solvents in steps (v) and(viii) can be the same or different.

The present invention also provides an extract obtainable by the processas described above, preferably from a plant extract, an Indigo Naturalisextract or an extract from the leaves and/or stems of an Indigo-bearingplant or Indigo-producing plant, preferably selected from the groupconsisting of Indigofera tinctoria L., Baphicacanthus cusia (Nees)Bremek (syn. Strobilanthes cusia (Nees), Persicaria tinctoria (Aiton)Spach. (syn. Polygonum tinctorium Aiton, P. tinctorium Lour.) and Isatistinctoria L. (syn. Isatis indigotica Fort.), said obtainable extractincludes indirubin as a component in an amount of at least 55% (w/w) ofthe refined extract, preferably at least 65% (w/w). The refined extractis in a solid form and may be prepared by the process described above orany other suitable process. The refined extract may further includeindigo, tryptanthrin, and/or qingdainone, which may be co-extractedtogether with indirubin. In particular, the extract containingmulti-ingredients may provide a synergistic effect.

The present invention further provides a pharmaceutical compositioncomprising the refined extract. The pharmaceutical composition may beformulated into a suitable dosage form for topical or oraladministration using technology well known to those skilled in the art.The pharmaceutical composition can additionally comprise apharmaceutically acceptable carrier such as those widely employed in theart of drug-manufacturing. For instance, the pharmaceutically acceptablecarrier may include one or more of the following agents: solvents,emulsifiers, suspending agents, decomposers, binding agents, excipients,stabilizing agents, chelating agents, diluents, gelling agents,preservatives, lubricants, absorption delaying agents, liposomes, andthe like. Preferably, solvents are selected from the group consisting ofwater, glycerol, ethanol, propylene glycol, butylene glycol, dipropyleneglycol, ethoxylated or propoxylateddiglycols, cyclic polyols, petroleumjelly, a vegetable oil and any mixture of these solvents. Preferably,the pharmaceutical composition is formulated into a topical formulationthat can be directly applied to the skin, for example, a skin sufferingfrom psoriasis. The topical formulation suitable for the pharmaceuticalcomposition may be an emulsion, a gel, an ointment, a cream, a patch, anembrocation, an aerosol, a spray, a lotion, a serum, a paste, a foam, ora drop. In one embodiment of the present invention, the pharmaceuticalcomposition is formulated into an external preparation by admixing therefined extract according to the present invention with a base such asthose that are well known and commonly used in the art.

In some embodiments, the dosage and the frequency of administration ofthe pharmaceutical composition according to the present invention mayvary depending on the following factors: the severity of the disease tobe treated, the route of administration, and the weight, age, physicalcondition and response of the subject to be treated. In further oradditional embodiments, the amount of the refined extract is in therange of about 0.001 to about 1000 mg/kg body weight/day, for example,about 0.01 to about 500, 300, or 100 mg/kg body weight/day. The presentinvention also provides a cosmetic composition comprising the refinedextract. The cosmetic composition may be present in a form adapted fortopical application comprising a cosmetically or dermatologicallyacceptable carrier or medium. “Cosmetically or dermatologicallyacceptable” means media which are suitable for a use in which they comeinto contact with the skin or human skin appendages without posing arisk of toxicity, intolerance, instability, allergic reaction, etc. Inthe cosmetic composition, the refined extract may be previouslysolubilized in one or more cosmetically or dermatologically acceptablesolvents, such as water, glycerol, ethanol, propylene glycol, butyleneglycol, dipropylene glycol, ethoxylated or propoxylateddiglycols, cyclicpolyols, petroleum jelly, a vegetable oil or any mixture of thesesolvents.

The refined extract and compositions above can be used in the treatmentor alleviation of a disease or condition. By treatment it is meant atleast an alleviation of the symptoms associated with the pathologicalcondition afflicting the subject, where alleviation is used in a broadsense to refer to at least a reduction in the magnitude of a parameter,e.g. symptom, associated with the pathological condition being treated,such as dermatitis, psoriasis and the like. As such, treatment alsoincludes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g., preventedfrom happening, or stopped, e.g., terminated, such that the host nolonger suffers from the pathological condition, or at least the symptomsthat characterize the pathological condition. As such, treatmentincludes both curing and managing a disease condition. Accordingly, theextract and compositions above can be used in the treatment oralleviation of a disease or condition selected from the group consistingof psoriasis, inflammatory skin conditions, onychomycosis, skin cancer,abnormal keratinization induced diseases, skin aging, andpustulardermatosis.

The present invention further provides a method for inhibitingproliferation or differentiation of keratinocytes, inhibitinginfiltration of mononuclear cells into the dermis and epidermis,inhibiting vascular alteration resulting in hyperplastic blood vessels,or inhibiting upregulation of adhesion molecules on endothelia cellscomprising administering the extract and compositions above to a subjectin need thereof.

The efficacy of the refined extract and compositions can be evaluated byin vitro models with respect to their activities in inhibitingproliferation or differentiation, or inflammation. For example, in vitrotesting can be conducted on cytokine signaling, STAT-3/STAT-1, MAPK,NFκB involved pathways.

The efficacy of the refined extract and compositions can be furtherevaluated by in vivo models with respect to their activities in treatingdiseases or disorders. For example, genetically engineered mice,including the transgenic and knockout models, can be tested.

The novel features of the application are set forth with particularityin the appended claims. A better understanding of the features andadvantages of the present application will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the application are utilized.

While preferred embodiments of the present application have been shownand described herein such embodiments are provided by way of exampleonly. It should be understood that various alternatives to theembodiments of the application described herein may be employed inpracticing the application. Those ordinary skilled in the art willappreciate that numerous variations, changes, and substitutions arepossible without departing from the application. It is intended that thefollowing claims define the scope of aspects of the application and thatmethods and structures within the scope of these claims and theirequivalents be covered thereby.

It is to be understood that, if any prior art publication is referred toherein, such reference does not constitute an admission that thepublication forms a part of the common general knowledge in the art. Alldocuments, or portions of documents, cited in the application including,without limitation, patents, patent applications, articles, books,manuals, and treatises are hereby expressly incorporated by reference intheir entirety for any purpose. The percentage herein is expressed byweight relative to the weight of the extract or the specified product,unless otherwise specified.

Further aspects and advantages of the invention will be disclosed in thefollowing illustrative experimental section.

EXAMPLES

1. Preparation of Refined Indigo Naturalis Extracts and AnalyticalMethods for Analysis

Example 1: Preparation of a Refined Indigo Naturalis Extract

Qingdai as used in the following preparation is obtained from DelongPharmaceutical (Indigo 2.62% and Indirubin 0.284% (HPLC method depictedin Example 8A) and tryptanthrin 0.0046% (HPLC method depicted in Example8B)).

500 g of Qingdai were suspended in 10 L ethyl acetate. The mixture wasstirred in reflux for two hours, and then filtered at 75° C. Thefiltrate was concentrated at reduced pressure to yield a dark solid. Thecrude extract was stirred in 250 mL hexane and heated to reflux for onehour. After cooling to room temperature, the suspension was filtered togive a dark residue (a crude extract).

0.50 g of the dark residue were refluxed in 25 mL hexane again for onehour, and cooled to room temperature, followed by filtration to give arefined extract as a dark red solid 452 mg. HPLC: 62.9% indirubin, 12.9%indigo, and 0.53% tryptanthrin.

Example 2: Preparation of a Refined Indigo Naturalis Extract

500 g of Qingdai as used in Example 1 were suspended in 10 L alcohol(ethanol). The mixture was stirred in reflux for two hours, and thenfiltered at 75° C. The filtrate was concentrated at reduced pressure toyield a dark solid, which was stirred in 260 mL hexane and heated toreflux for one hour. Upon cooling to room temperature, the suspensionwas filtered to give a dark residue.

0.80 g of the dark residue were refluxed in 24 mL alcohol (ethanol) foran additional hour, and then cooled to room temperature, followed byfiltration to give a refined extract as a dark red solid (538 mg). HPLC:83.6% indirubin, 6.35% indigo, and 0.75% tryptanthrin.

Example 3: Preparation of a Refined Indigo Naturalis Extract

500 g of Qingdai as used in Example 1 were suspended in 10 L ethylacetate. The mixture was stirred in reflux for two hours, and thenfiltered while hot. The filtrate was concentrated at reduced pressure toyield a dark solid. The crude extract was stirred in 250 mL hexane andheated to reflux for one hour. After cooling to room temperature, thesuspension was filtered to give a dark residue.

0.75 g of the dark residue were refluxed in 22.5 mL ethanol for onehour, and cooled to room temperature, followed by filtration to give arefined extract as a dark red solid (538 mg). HPLC: 77.9% indirubin,15.9% indigo, and 0.56% tryptanthrin.

Example 4: Preparation of a Refined Indigo Naturalis Extract

500 g of Qingdai as used in Example 1 were suspended in 2.1 L DMF. Themixture was stirred at 50° C. for 40 minutes. Upon cooling to 20° C.,the suspension was filtered. The filtrate was concentrated at reducedpressure to yield a dark solid, which was stirred in 130 mL hexane andheated to reflux for one hour. Upon cooling to 20° C., the suspensionwas filtered to give a dark residue.

1.56 g of the dark residue was washed with 46.8 ml ethanol, and heatedto reflux for one hour, and then cooled to 20° C., followed by filteredto yield a refined extract (766 mg). HPLC: 66.3%, indirubin, 9.76%indigo.

Example 5: Preparation of a Refined Indigo Naturalis Extract

500 g of Qingdai as used in Example 1 were suspended in 3 L DMF. Themixture was stirred at 30° C. for 1 hour, and then filtered. Thefiltrate was concentrated at reduced pressure to yield a dark solid,which was stirred in 230 mL hexane and heated to reflux for one hour.Upon cooling to 20° C., the suspension was filtered to give a darkresidue.

1.96 g of the dark residue was washed with 59 mL 85% ethanol (85% aq.alcohol), and heated to reflux for one hour followed by filtration whilehot to yield a refined extract (1.02 g). HPLC: 69.4% indirubin, 18.7%indigo, and 0.62% tryptanthrin.

Example 6: Preparation of a Refined Indigo Naturalis Extract

100 g of Qingdai was extracted with 2 L of ethanol 92% (92% aqueousethanol) for 2 hours under reflux conditions. Upon completion, themixture was filtered while hot on AF6 filter (Buchner) to obtain a darkblue-red solution as a filtrate. This filtrate was reduced under vacuumto dryness to give 2.4 g of dry residue. This residue was washed with120 mL of hexane for 1 h under reflux. Upon completion, the mixture wascooled to room temperature for 2 h then filtered under vacuum to yield312.9 mg of a dark red refined extract.

280 mg of this refined extract were washed with 15 mL of ethanol 92%(92% aqueous ethanol) for 1 h under reflux. Upon completion the solutionwas cooled to room temperature, and then filtered to yield 159 mg of adark red/burgundy refined extract after drying in oven (80° C.) for 1h30. (0.18%); HPLC: 82.31% indirubin, 8.99% indigo, and 0.81%tryptanthrin.

Example 7: Micronization Step

The micronization step of refined Indigo Naturalis extract obtained inthe previous examples is performed with the following equipment:

-   -   Micronizer: spiral jet Mill Diameter 200    -   Feeder: this equipment is used for the dosage of powder to feed        the micronizer. The dosage is made thanks to two screws. This        system allows a regularity of the flow.

Micronization consists to project grains of powder with jet of air. Thecontact of grains permits their explosion.

Following parameters of micronization are recorded during themicronization:

-   -   Ring pressure: 6 bar    -   Injector pressure: 3 bar    -   The flow of powder feed: 25 kg/h

The micronizer allows a cylindrical enclosure—holes around the enclosurefor the injection of air.

Powder is introduced in the micronizer; grains are propelled thanks tojet of air. When grains have the good size, they are concentrated in thecenter of the micronizer and they are breathed. To avoid anycontamination by foreign particles or broken pieces of the equipment, anadditional sieving (sieve: 700 μm) is performed.

The step is done manually after the micronization and before thepackaging.

A granulometric analysis of the homogeneous product obtained was carriedout according to the particular size distribution (PSD) method[Analytical specifications: D99≤30 μm].

Example 8: Analytical Methods for Analysis

A—Reversed phase HPLC method:

A new reversed phase HPLC method to quantitate indigo and indirubinsimultaneously was established based on European Pharmacopoeia(Pharmeuropa Vol. 20, No. 1, January 2008, P118-119.), ChinesePharmacopoeia (2010 Edition, P185) and literatures (Chen L W, Liao W,Yang M. Jia D Y, He P, Chen S M, Fu C M.

Determination of indigo and indirubin in Indigo Naturalis by HPLC. WestChina Journal of Pharmaceutical Sciences, 2008, 23(6), 714-715; Liu Z Y,Su Z T, Gao Y N, Yang M. Simultaneously determination of indigo andindirubin in Indigo Naturalis by HPLC. China Pharmacist 2010, 13(3),324-326).

The chromatographic system (Agilent 1200 series) consisted of a G1322Adegasser, a G1211A pump, a G1367B autosampler, a G1316A column oven anda G1315B DAD detector. Other apparatus included a SK7200H ultrasonicdevice (China) and a Milli-Q water purification system (USA).

Six batches of Qingdai were collected from three vendors in China.Indigo standard was purchased from Tokyo Chemical Industry Co.(Japan, >98%). Tryptanthrin was bought from Accela Co. (China, 97%).Indirubin was synthesized and recrystallized at Hutchison Medipharma(HMP) (>99% in HPLC).

Organic filter membrane (0.45 μm, China), Dimethyl formamide (DMF,analytical grade), methanol (HPLC grade), formic acid (FA, HPLC grade),triethylamine (TEA, analytical grade) and ultra-pure water purified withMilli-Q water purification system were used in the experiments.

Pretreatment of DMF solution: 500 mL of DMF was blown with dry N2 forhalf an hour, then 0.5 mL TEA was added and mixed to give a DMF solution(containing 0.1% TEA, free of oxygen). This DMF solution was used insample preparation.

50 mg of Qingdai was suspended in 50 mL DMF solution. After ultrasonicextraction for 10 min, the suspension was filtered through 0.45 μmsyringe filter to generate the test solution of Qingdai.

20 mg of refined extract (obtained from Example 2) was suspended in 200mL DMF solution. After ultrasonic extraction, the suspension wasfiltered through a 0.45 μm syringe filter to generate the test solutionof the extract.

The separation was performed on a Waters Symmetry C18 column (5 μm,3.9×150 mm). The mobile phase was 65% methanol (containing 0.05% FA).The flow rate was 1.0 mL/min for 15 min and the column temperature was25° C. Injection volume was 4 μL. Detection wavelength was 289 nm sothat indigo and indirubin could be assayed simultaneously. Indigo andindirubin could be analyzed simultaneously in one injection. TypicalHPLC chromatograms of indigo and indirubin were shown in FIGS. 1A and1B.

B—HPLC analytical method to quantitate tryptanthrin:

A new HPLC analytical method to quantitate tryptanthrin was alsoestablished. The sample concentration would be adjusted accordingly dueto the low concentration of Tryptanthrin in both Qingdai and itsenriched product, the refined extract. The analyses were performed at25° C. on a Waters Symmetry C18 column (5 μm, 3.9×150 mm). The mobilephase was methanol (containing 0.05% FA, eluent B) and water (containing0.05% FA, eluent A). The gradient elution profile was as follows: 50% Bisocratic (12 min), 50% to 100% B (1 min), 100% B isocratic (6 min) and100% to 50% B (1 min). The flow rate was 1.0 mL/min and the columntemperature was 25° C. Injection volume was 10 μL. Detection wavelengthwas 254 nm.

400 mg of Qingdai was suspended in 20 mL DMF solution. After ultrasonicextraction for 20 min, the suspension was filtered through 0.45 μmsyringe filter to provide the test solution of Qingdai.

15 mg of refined extract (obtained from Example 2) was suspended in 20mL DMF solution. After ultrasonic extraction, the suspension wasfiltered through 0.45 μm syringe filter to provide the test solution ofrefined extract.

Typical HPLC chromatograms of thryptanthrin, Qingdai and a refinedextract were shown in FIGS. 2A to 2C.

Example 9: Long Term Stability of a Refined Indigo Naturalis Extract

A refined Indigo Naturalis extract prepared according to example 6 hasbeen submitted to stability conditions. The HPLC characterization of theprepared extract is as follows: 80.16% indirubin; 10.95% indigo; 0.64%tryptanthrin.

The storage conditions used during stability study are 25° C.±2° C. and60% RH±5% RH in a climatic room (Piardi CC1400).

According to the GUIDELINE ON QUALITY OF HERBAL MEDICINAL PRODUCTS(CPMP/QWP/2819/00 rev 01), qualitative attributes have to comply withthe limits [Limits: content of indirubin (% dried extract)=65.0-85.0;content of indigo (% dried extract)=5.0-15.0; content of tryptanthrin (%dried extract)≤5.0.

Variations in content of markers have not to exceed±10% of the initialassay values.

Results are shown in the following table 1.

TABLE 1 T0 1 month 3 months 6 months % of % of % of % of Content initialContent initial Content initial Content initial (% dried assay (% driedassay (% dried assay (% dried assay Markers extract) value extract)value extract) value extract) value Tryptanthrin 0.64 100 0.64 100 0.65101 0.64 100 Indigo 10.95 100 11.03 101 11.10 101 11.12 102 Indirubin80.16 100 79.19 99 79.52 99 80.17 100 HPLC Fingerprint Complies CompliesComplies

HPLC fingerprint at 1 month, 2 months and 3 months complies withfingerprint chromatogram at T0. Thus, qualitative attributes comply atthe limits. Variation of content of Indigo, Indirubin and Tryptanthrindo not exceed the acceptable range of ±10% of the initial assay values.

Thus, a refined Indigo Naturalis extract prepared according to theinvention is stable at least 6 months at 25° C. and 60% RH.

2. In vitro Evaluation of Refined Indigo Naturalis Extracts inBiochemical and Cellular Assays

Example 10: In Vitro Assays and Results

A. General Reagents:

DMSO, Sigma-Aldrich, St. Louis, Mo., Cat. No. D2650

Janus kinase 1(JAK1), Life Technologies™, Cat. No. PV4774

Janus kinase 1(JAK2), Life Technologies™, Cat. No. PV4210

Janus kinase 1(JAK3), Life Technologies™, Cat. No. PV3855

CDK1, Life Technologies™, Cat. No. PV3292

CDK2, Life Technologies™, Cat. No. PV3267

CDK5, Life Technologies™, Cat. No. PV3000

Z′-LYTE® Kinase Assay Kit—Tyrosine 6 Peptide, Life Technologies™, Cat.No. PV4122

Z′-LYTE® Kinase Assay Kit—Ser/Thr 12 Peptide, Life Technologies™, Cat.No. PV3673

Dulbecco's modified Eagle's medium (DMEM), Life Technologies™, Cat. No.C11965

RMPI-1640, Life Technologies™, Cat. No. A10491

Fetal bovine serum (FBS), Life Technologies™, Cat. No. 10099141

EpiLife® Medium, Life Technologies™, Cat. No. M-EPI-500-CA

HKGS, Life Technologies™, Cat. No. S-001-5

Recombinant human IL-2, Peprotech Inc, Cat. No. 200-02

Recombinant human IL-6, Peprotech Inc, Cat. No. 200-06

Recombinant human IL-3, Peprotech Inc, Cat. No. 200-03

Recombinant human GM-CSF, Peprotech Inc, Cat. No. 300-03

Recombinant human IL-22, Peprotech Inc, Cat. No. 200-22

Recombinant human TNFα, R&D, Cat. No. 210-TA-010

Lipopolysaccharide (LPS), Calbiochem, Cat. No. 437650

Anti-Human CD3 Functional Grade® Purified (aCD3) (Clone: OKT3)eBioscience,

Cat. No. 16-0037-85

Anti-Human CD28 Functional Grade® Purified (aCD28) (Clone: CD28.2),

eBioscience, Cat. No. 16-02897-85

phospo-STAT3 (Y705) antibody (rabbit-anti-human), Cell SignalingTechnology, Cat. No. 9145

phospo-STATS (Y694) antibody (rabbit-anti-human), Cell SignalingTechnology, Cat. No. 9359

Actin antibody (mouse-anti-human) Sigma-Aldrich, Cat. No. A1978

Goat anti-rabbit IgGAlexa 488, Life Technologies™, Cat. No. A11034

Goat-anti-rabbit IROYE 800CW, Li—COR Bioscience, Cat. No. 926-32211

Goat-anti-mouse IROYE 800CW, Li—COR Bioscience, Cat. No. 926-32210

Human IFNγ ELISA Kit, R&D, Cat. No. DY285

Human TNFα ELISA Kit, R&D, Cat. No. DY210

Human IL-1β ELISA Kit, R&D, Cat. No. DY201

Human IL-6 ELISA Kit, R&D, Cat. No. DY206

Thiazolyl Blue Tetrazolium Blue (MTT), Sigma-Aldrich, Cat. No. M2128

CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Cat. No. G7572

CytoTox-ONE™ Homogeneous Membrane Integrity Assay, Promega, Cat. No.G7891

Luciferase Assay, Promega, Cat. No. E4550

iBlot® Transfer Stack, Regular (Nitrocellulose), Life Technologies™,Cat. No.

IB3010-01

Propidium iodide, Sigma-Aldrich, Cat. No. P4170

Ribonuclease A, Sigma-Aldrich, Cat. No. R6513

1×PBS Buffer (1 L): NaCl 8.0 g, KCl 0.2 g, Na₂HPO₄-12H₂O 3.58 g, KH₂PO₄0.24 g, dissolved in 1 L MilliQ ddH₂O, pH adjusted to 7.4.

1×SDS loading buffer: 50 mMTris-HCl/pH8.8, 2% SDS, 10% glycerol, 0.1%bromophenol blue, 100 mM DTT.

B. Cell and Cell Lines

HepG2, a human hepatocellular carcinoma cell line, purchased fromShanghai Institutes for Biological Sciences (SIBS) (Shanghai, China,Cat. No. TCHu 72), were cultured in DMEM containing 10% FBS.

TF1, a human erythroleukemia cell line, purchased from American TissueCulture Collection (ATCC) (Manassas, Va., Cat. No. CRL-2003™), werecultured in RMPI-1640 containing 10% FBS and 2 ng/mL of GM-CSF at 37° C.with 5% CO₂.

PBMCs: Normal human blood samples from healthy adult donors werecollected in heparinized tubes. Each independent experiment used bloodfrom a single healthy donor. Mononuclear cells (PBMCs) were isolatedusing Ficoll-Paque Plus reagent (Amersham Pharmacia Biotech, Sweden,Cat. No. 17-1440-02) according to protocol recommended by themanufacturer and cultured in RMPI-1640 containing 10% FBS at 37° C. with5% CO₂.Primary T cells: Mononuclear cells (PBMC) were isolated usingFicoll-Paque Plus reagent (Amersham Pharmacia Biotech, Sweden, Cat. No.17-1440-02) according to protocol recommended by the manufacturer. Thencells were activated by using anti-CD3 (1 μg/mL) and anti-CD28 (5 μg/mL)for 3 days, and expanded in RMPI-1640 containing 10% FBS and long/mLIL-2 at 37° C. with 5% CO₂ every 2-3 days for 2 weeks prior toperforming experiments.HaCaT, a human epidermal keratinocyte line from The Second MilitaryMedical University (SMMU), China, was cultured in DMEM containing 10%FBS.HEKa, human epidermal keratinocytes isolated from adult skin, purchasedfrom Life Technologies™ (Carlsbad, Calif., USA, Cat. No. C-005-5C) andcultured in EpiLife 0 Medium containing HKGS at 37° C. with 5% CO₂.293/NFkB-Luc cell line was purchased from Panomics (Fremont, Calif.,Cat. No. RC0014). It was obtained by co-transfection with pNFκB-Luc andpHyg into a human embryonic kidney 293 cells, followed by hygromycinselection. The cells were cultured in DMEM containing 10% FBS and 100μg/mL hygromycin B (Life Technologies™, Cat. No. 10687-010).

C. Kinase Assay

JAK1/2/3 kinase assays were performed in vitro using recombinant human

JAK1/2/3 and Z′-LYTE® Kinase Assay Kit—Tyrosine 6 Peptide. CDK1/2/5kinase assays were performed in vitro using recombinant human CDK1/2/5and Z′-LYTE® Kinase Assay Kit—Ser/Thr 12 Peptide. All reactions (20 μL)were started by adding 2.5 μL of positive control (CP-690550 for JAKkinase assay and Staurosporine for CDK kinase assay) or the testarticles (i.e., samples) in 4% DMSO solution, 5 μl of Kinase/Peptidesubstrate Mixture or Phospho-Peptide solution, 2.5 μL ATP Solution (100μM) or 1.33× Kinase Buffer. The 384-well assay plate (Corning, Cat. No.3575) was mixed and incubated at room temperature for 1 hour. 5 μL ofthe Development Solution was then added to each well, mixed andincubated at room temperature for another 1 hour. The kinase reactionswere then stopped by adding 5 μL of the Stop Reagent followed byrecording 450 nm and 520 nm fluorescence's using Perkin-Elmer Victor III(Perkin-Elmer Life Sciences, Boston, Mass.) plate reader.

D. Acumen Assay

For IL-6 induced STAT3 phosphorylation, HepG2 were seeded in 96 wellplates at 5.4×10³ cells per well in serum-free DMEM media at 37° C., 5%CO₂ overnight. After incubation with CP-690550 or test articles for 30minutes, cells were stimulated by adding 100 ng/mL human recombinantIL-6 (1:10) to each well for 15 minutes.

For IL-3 induced STATS phosphorylation, TF-1 was seeded in 96-wellplates at 1×10⁴ cells per well at 37° C., 5% CO₂ for 3 hours. Afterincubation with CP-690550 or test articles for 30 minutes, cells werestimulated by adding 100 ng/mL human recombinant IL-3(1:10) to each wellfor 30 minutes.

HepG2 or TF1 cells were then fixed in 2% paraformaldehyde for 45 minutesat room temperature and incubated in ice-cold methanol for 30 minutes.After washing with PBS, cells were incubated with anti-phospho-STAT3(Y705) or anti-phospho-STATS (Y694) antibody respectively at 4° C.overnight. Goat anti-rabbit IgG Alexa 488 secondary antibody was addedfor 90 minutes prior to PBS washes. Cells were counted followingincubation in PBS containing 7.5 μM Propidium iodide and 100 μg/mLRibonuclease A for 60 minutes in the dark. Plate was read on an AcumenX3 instrument (TPP Labtech, Hertfordshire SGB, UK).

E. Western Blots

HEKa were seeded in 6-well plates at 2×10⁵ cells/well at 37° C., 5% CO₂overnight. After incubation with test article for 30 minutes, the cellswere stimulated with 100 ng/mL IL-22 for 30 minutes.

After the treatment, samples were collected in 1×SDS loading buffer.Protein samples were boiled for 15 minutes and collected bycentrifugation at 14,000 g for 10 min at 4° C. The supernatants wereused or immediately stored at −80° C. For Western blot analysis, sampleswere separated on a 10% Tris-HCL gradient electrophoresis gel (Bio-RadLaboratories). Gels were blotted onto a iBlot® Transfer Stack, Regular(Nitrocellulose) which was blocked in 5% nonfat dry milk and probedusinganti-phospho-STAT3 (Y705) antibody or anti-Actin antibody at 4° C.overnight, respectively. The membrane was then incubated withappropriate IDRye 800CW secondary antibody followed by detection usingOdyssey infrared imaging System (Li—COR Bioscience, Lincoln, Nebr.,USA).

F. Reporter Assays

For reporter gene assays, the 293/NFκB-Luc was seeded in 96-well plateat 4×10⁴ cells per well overnight. After the incubation withAndrographolide (LGT) or test articles for 30 minutes, cells werestimulated by adding 100 ng/mL TNFα (1:10) to each well for 6 hours.Cell lysates were prepared by removing media and adding lysis buffer. 5×volume Luciferase Assay Reagent was added to each well prior to readplate. Luminescence was recorded using Perkin-Elmer Victor III platereader (Perkin-ElmerLife Sciences, Boston, Mass., USA).

G. ELISA Assay

Primary T cells were seeded in 96-well plates at 8×10⁴ cells/well. Testarticles were added into the cultures and incubated at 37° C., 5% CO₂.After 30 minutes, the cell suspension in each well was transferred toanother 96-well plate coated with a CD3 (1 μg/mL) and a CD28 (5 μg/mL)and incubated at 37° C. with 5% CO₂ for 22 hours. The media were removedand stored at −80° C. until assayed. IFNγ concentrations were determinedusing a commercial ELISA kit (R & D Systems), following themanufacturer's instruction.

PBMCs were seeded in 96-well plates at 3×10⁴ cells per well. Testarticles were then added into the culture and incubated at 37° C., 5%CO₂. After 30 minutes, 1 μg/mL LPS (1:10) was added to the culture. Forquantitation of protein levels, the plates were incubated for 18 hours.The media were removed and stored at −80° C. until assayed. TNFα, IL-βand IL-6 concentrations were determined using commercial ELISA kits (R &D Systems), following the manufacturer's instructions.

H MTT Assay

HaCaT were seeded in 96-well plates at 4×10⁴ cells/well overnight.Stausporine or test articles were then added into the culture andincubated at 37° C., 5% CO₂ for 72 hours. After removal of the media,the cells in 96-well plates were exposed to 100 μL of MTT (0.5 mg/mL inDMEM containing 10% FBS per well and incubated for 3 hours at 37° C., 5%CO₂. Subsequently, the supernatants were removed and 150 μL of DMSO wereadded to each well. The plate was incubated in the dark for 10 minutesand the absorbance at 492 nm was recorded immediately using MultiskanMK3microplate reader (Thermo Life Sciences, HK, China).

I. Cell Viability Assay

CellTiter-Glo® Luminescent Cell Viability Assay Kit was used toinvestigate cell viability. HEKa were seeded in 96-well opaque-walledplate at 1×10⁴ cells/well overnight. Dithranol or test articles werethen added into the culture and incubated at 37° C., 5% CO₂ for 48hours. Cells were lysed with CellTiter-Glo® Reagent equal to the volumeof cell culture media present in each well, and contents mixed for 2minutes to induce cell lysis. Plate was incubated at room temperaturefor 10 minutes to stabilize luminescent signal. Luminescence wasrecorded using Perkin-ElmerVictor III plate reader (Perkin-Elmer LifeSciences, Boston, Mass., USA).

J. LDH Release Assay

Lactate dehydrogenase (LDH) release assay kit was used to investigatecytotoxicity. HEKa were seeded in 96-well opaque-walled plates at 4×10⁴cells/well overnight. Dithranol or test articles were then added intothe culture and incubated at 37° C. under 5% CO₂ for 24 hours.Supernatants and cell lysates were prepared. 1× volume of CytoTox-ONE™Reagent equal to the volume of supernatants or cell lysates were addedto each well followed by mixing for 30 seconds and incubating at 25° C.for 10 minutes. Stop solution equal to 50% volume of the supernatants orcell lysates were added to each well to stop the reaction, andfluorescence with an excitation wavelength of 560 nm and an emissionwavelength of 590 nm were recorded using SpectraMax M2 (MolecularDevices, Sunnyvale, Calif., USA).

K. TNFα-Induced NFκB Activation

Pro-inflammatory cytokines and chemokines play important roles inpathogenesis of psoriasis. NFκB is clearly one of the most importantregulators of pro-inflammatory cytokine and chemokine gene expression.Therefore, inhibitory potency of Qing Dai and its refined extracts onTNFα-dependent NFκB activation was investigated in HEK 293/NFκB-Luc. Asshown in Table 1, TNFα stimulated NFκB-dependent luciferase expressionand pretreating cells with Tripterygium Glycoside blocked NFκBactivation in a concentration dependent manner. Indigo

Naturalis refined extract had microgram/g activities on TNFα-dependentNFκB activation on the experimental condition. (see Table 1).

L. IC₅₀ Determinations

All IC50 values were determined by using Xlfit™ software (version 2.0)from ID

Business Solutions (Guildford, UK). Background was defined in culturewith cells treated with DMSO only and was subtracted for IC₅₀calculations.

M. Results

Some in vitro assay results of Indigo Naturalis refined extracts areshown in Table 2 below, wherein Indigo Naturalis A is obtained fromDelong Pharmaceutical: (Indigo 2.62%, Indirubin 0.284%; Tryptanthrin0.0046%).

TABLE 2 pro-inflam- matory cytokine HaCaT HEKa production proliferationViability IC₅₀ (NFκB) IC₅₀ IC₅₀ Sample μg/mL μg/mL μg/mL Staurosporine3.18(nM) +/− 2.19 LGT 0.00045 Indigo Naturalis A** 2.42/2.43 Example 410.87/63.53/ 1.57/<0.82/1.02 10.37/25.75 Example 1  8.47/15.40 2.91 ±0.49 (n = 2) Example 3 37.23/>100  1.71 ± 0.54 (n = 3) Example 251.03/41.79 1.75 ± 0.48 (n = 4) Example 5 24.66/32.97 2.21 ± 0.45 (n =4) **Indigo Naturalis A: Indigo Naturalis from Delong Pharmaceutical:Indigo 2.62%, Indirubin 0.284%; Tryptanthrin 0.0046%3. In Vivo Evaluation of Refined Indigo Naturalis Extracts in AnimalModels

Example 11: In Vivo Assays and Results

A. Materials and Methods

Animals

BABL/c mice, male, body weight 19-22 g, purchased from Shanghai SLCAnimalCenter.

Room temperature: 24±1° C.

Room relative humidity: 40-70%

Light cycle: Fluorescent light for 12-hour light (8:00-20:00) 12-hourdark

Animal hosting: 4 mice/cage

Food: Free access to food (irradiated, Shanghai SLAC Laboratory AnimalCo. Ltd., China)

Water: Free access to tap water from local supply (first filtered byMolanimal ultrapure water machine from the municipal water supply)

Instruments

MJ Research PTC-200 Peltier Thermal cycler (Alpha Unit™ Block Assemblyfor PTC DNA Engine™ systems)

Applied Biosystems 7500 realtime PCR system

Digimatic micrometer caliper: Mitutoyo, Japan. Accuracy: 0.001 mm

Reagents

Recombinant mouse IL-22 (rmlL-22), Novoprotein (sinobio), Cat. C047,Lot. 0375351

High capacity cDNA Reverse Transcription kit, Applied Biosystems, PartNo.:4368813, Lot: 0909069

Thermo Scientific ABsolute SYBR Green Rox Mix, Thermo Scientific, Cat:

AB-1163/A, Lot: 0911/16

Positive Control

Protopic® (tacrolimus, FK506), 0.1% ointment, Astellas Toyama Co., Ltd.Toyama Plant, H20100079, Lot: 028680.

Dosing Regimen

Different concentrations of test samples, vehicle, or 0.1% of FK506ointment, were topically administrated at 1% one hour after theinduction of model and then given daily from day 1 to day 11, twice aday. The first day of administration of a test article was regarded asday 0.

Route of Administration

Topical application, b.i.d.

Establishment of IL-22 Induced Psoriasis-Like Mouse Models

Intradermal injection of 20 μL PBS, either alone or containing 100 or500 ng recombinant mouse IL-22 (eBiosience), was administered into theears of anesthetized mice using a 30-gauge needle every other day foreleven days. Ear thicknesses were measured before injection on day 0 andthereafter on days without injections. Ear measurements were taken atthe center of the ears using a digimatic micrometer caliper (Mitutoyo).

Twenty-four hours after the last IL-22 treatment, mice were sacrificed,and ears were collected for further analysis.

Histological Examination

Ears were collected at necropsy, fixed in 10% buffered formalinphosphate, embedded in paraffin, sectioned, and stained withhematoxylin/eosin (H&E). Microscopic sections were graded by the numberand severity of lesions.

Statistical Methods

Results of ear thickness data were expressed as mean±S.E.M. AUC of earswelling was calculated by the ear thickness data from day 0 to day 11,and analyzed by repeated-measured ANOVA methods with JMP® software.Cytokine protein and gene expression data were evaluated with a one-wayANOVA and followed by student's t-test for post-hoc analysis.(Significance level was set at p<0.05).

Group and Dosing

See FIG. 3.

Results

Some in vivo assay results of some of Indigo Naturalis refined extractsare shown in Table 3.

TABLE 3 inhibition of In vivo ear thickness on day 11 AUC of ear AUC ofear studies group (μm) thickness thickness (%) study 1 saline-control207.8 ± 1.8, 202.8 ± 2.2 61 (cf FIG. 4) (naïve) (d 15) IL-22, 100 ng248.3 ± 1.9, 255.3 ± 15.1 590 (d 15) IL-22, 500 ng 311.3 ± 18.0, 296.3 ±5.0 1055 study 2 naïve control 236.7 ± 2.8 172 vehicle 1 control 371.3 ±8.7 1281 FK506 (0.1%) 256.8 ± 3.5 403 79.2 Indigo Naturalis B* (10%) 328.8 ± 16.8 749 48 study 3 naïve control 230.2 ± 4.4 165 (cf FIG.5(A)) vehicle control 428.3 ± 6.6 1256 FK506 (0.1%) 278.8 ± 5.0 369 81.3Indigo Naturalis B* (10%)  349.3 ± 14.6 703 50.7 Example 4 (1%)  289.3 ±17.4 876 34.8 Example 4 (0.1%)  408.7 ± 18.0 975 25.7 study 4 naïvecontrol 233.1 ± 3.6 238 vehicle control  356.4 ± 11.8 1301 FK506 (0.1%)260.4 ± 4.5 446 80.4 Example 4 (0.5%) 310.4 ± 4.4 969 31.3 Example 4(0.1%) 303.8 ± 4.3 868 40.7 Example 4 (0.02%) 302.8 ± 6.4 922 35.7 study5 naïve control 214.5 ± 1.1 143 (cf FIG. 5(B)) vehicle control 334.3 ±3.3 1194 FK506 (0.1%) 263.4 ± 6.3 454 70.4 Example 3 (0.02%) 289.6 ± 8.0751 42.2 Example 2 (0.02%) 276.3 ± 3.2 627 53.9 Example 5 (0.02%) 275.5± 5.9 677 49.2 *Indigo Naturalis B: Indigo Naturalis from QingfengPharmaceutical: Indigo 2.02%, Indirubin 0.216%; Tryptanthrin 0.0032%

The invention claimed is:
 1. An extract product of Indigo Naturalisprepared by a process comprising: a) extracting the Indigo Naturaliswith a solvent to obtain a mixture of extraction, wherein the solvent isselected from the group consisting of dimethylformamide, ethyl acetate,ethanol, dimethylsulfoxide, dichloromethane, tetrahydrofuran,dimethylacetamide, acetone, 2-butanone, acetonitrile, isopropyl acetate,2-methyl tetrahydrofuran, methyl tert-butyl ether, methanol, chloroform,terpene, water and a combination thereof, b) filtering the mixture ofextraction to obtain a filtrate, c) concentrating the filtrate to obtaina crude extract, d) mixing the crude extract and hexane or heptane inreflux to obtain a first washing mixture, e) cooling and filtering thefirst washing mixture at room temperature to obtain a first extractproduct, and f) washing the first extract product with a washing methodcomprising: i. mixing the first extract product and a solvent selectedfrom the group consisting of hexane, ethanol and an aqueous solution ofethanol in reflux to obtain a second washing mixture, and ii. coolingand filtering the second washing mixture at room temperature to obtainthe extract product of Indigo Naturalis.
 2. The extract product ofIndigo Naturalis of claim 1, wherein in a), the solvent is selected fromthe group consisting of dimethylformamide, ethyl acetate, ethanol, and acombination thereof, or an aqueous solution thereof.
 3. The extractproduct of Indigo Naturalis of claim 2, wherein in a), the solvent isethyl acetate, ethanol or an aqueous solution of ethanol, and themixture of extraction is obtained by mixing the Indigo Naturalis and thesolvent in reflux.
 4. The extract product of Indigo Naturalis of claim3, wherein in b), the mixture of extraction is filtered at a temperaturenot less than 65° C. to obtain the filtrate.
 5. The extract product ofIndigo Naturalis of claim 4, wherein prior to d), the crude extract isadded with water and filtered to remove the solvent in the crudeextract.
 6. The extract product of Indigo Naturalis of claim 5, whereinin d), the crude extract is mixed with hexane in reflux to obtain thefirst washing mixture.
 7. The extract product of Indigo Naturalis ofclaim 6, wherein the first extract product is rinsed with ethanol andwater prior to f).
 8. A refined extract of Indigo Naturalis prepared bya process comprising: a) extracting the Indigo Naturalis with a solventto obtain a mixture of extraction, wherein the solvent is selected fromthe group consisting of ethyl acetate, ethanol, an aqueous solution ofethanol, and a combination thereof, and the mixture of extraction isobtained by mixing the Indigo Naturalis and the solvent in reflux, b)filtering the mixture of extraction at a temperature of not less than65° C. to obtain a filtrate, c) concentrating the filtrate to obtain acrude extract, d) mixing the crude extract and hexane in reflux toobtain a first washing mixture, e) cooling and filtering the firstwashing mixture at room temperature to obtain a first extract product,and f) washing the first extract product with a washing methodcomprising: a) mixing the first extract product and a solvent selectedfrom the group consisting of hexane, ethanol and an aqueous solution ofethanol in reflux to obtain a second washing mixture, b) cooling andfiltering the second washing mixture at room temperature to obtain asecond extract product, and c) drying the second extract product at atemperature of less than 80° C. to obtain a dried extract product, g)optionally washing the dried extract product with the washing method off) one or more times to thereby obtain the refined extract, wherein, therefined extract of Indigo Naturalis comprises at least 55% (w/w)indirubin relative to the total weight of the refined extract.
 9. Therefined extract of Indigo Naturalis of claim 8, wherein the processfurther comprises micronizing the fine extract.
 10. A pharmaceuticalcomposition comprising the extract product of Indigo Naturalis of claim1 and a pharmaceutically acceptable carrier.
 11. A pharmaceuticalcomposition comprising the refined extract of Indigo Naturalis of claim8 and a pharmaceutically acceptable carrier.
 12. A pharmaceuticalcomposition comprising the refined extract of Indigo Naturalis of claim9 and a pharmaceutically acceptable carrier.
 13. A method of treating adisease or condition selected from the group consisting of psoriasis,inflammatory skin conditions, onychomycosis, skin cancer, abnormalkeratinization induced diseases, skin aging, pustulardermatosis, andCTCL in a subject in need thereof, comprising administering to thesubject the pharmaceutical composition of claim
 10. 14. The method oftreating the disease or condition according to claim 13, wherein thedisease or condition is psoriasis.
 15. A method of treating a disease orcondition selected from the group consisting of psoriasis, inflammatoryskin conditions, onychomycosis, skin cancer, abnormal keratinizationinduced diseases, skin aging, pustulardermatosis, and CTCL in a subjectin need thereof, comprising administering to the subject thepharmaceutical composition of claim
 11. 16. The method of treating thedisease or condition according to claim 15, wherein the disease orcondition is psoriasis.
 17. The method of treating the disease orcondition according to claim 16, wherein the psoriasis is one or moreselected from the group consisting of chronic plaque psoriasis, guttatepsoriasis, erythrodermic psoriasis, pustular psoriasis, inversepsoriasis, nail psoriasis, and psoriatic arthritis.
 18. A method oftreating a disease or condition selected from the group consisting ofpsoriasis, inflammatory skin conditions, onychomycosis, skin cancer,abnormal keratinization induced diseases, skin aging,pustulardermatosis, and CTCL in a subject in need thereof, comprisingadministering to the subject the pharmaceutical composition of claim 12.19. The method of treating the disease or condition according to claim18, wherein the disease or condition is psoriasis.
 20. A method oftreating the disease or condition according to claim 19, wherein thepsoriasis is one or more selected from the group consisting of chronicplaque psoriasis, guttate psoriasis, erythrodermic psoriasis, pustularpsoriasis, inverse psoriasis, nail psoriasis, and psoriatic arthritis.